First, Definitions:
During the description of the invention will be used a terminology whose meaning is listed bellow.
By CTXΦ virus is meant the particle of protein-coated DNA produced by certain V. cholerae strains, which is capable of transducing its DNA, comprising cholera toxin genes, to other vibrios.
By cholera toxin (CT) is meant the protein responsible for the clinical symptoms of cholera when produced by the bacteria.
By CTXΦ-encoded toxin genes are meant, in addition to CT genes, zot and ace genes that encode for the “zonula occludens toxin” and for the accessory cholera enterotoxin, respectively. The activity of ZOT is responsible for the destruction of the tight junctions between basolateral membranes of the epithelial cells and ACE protein has an activity accessory to that of the cholera toxin.
The term well tolerated vaccine or well tolerated strain refers to such strain lacking the residual reactogenicity that characterize most of the of non-toxigenic strains of V. cholerae. In practical terms, it means that it is a strain safely enough to be used in communities without or with limited access to healthcare institutions without risks for the life of the vaccinees. It should be expected a rate of diarrhea in 8% or less of the vaccinees and the diarrhea is characterized in that it does not exceed 600 ml (grs), only 1% of the vaccinees or less could suffer from headache, which should be minor and of short duration (less than 6 h), and finally that it prompts vomits in less than 0.1% of the vaccinees, those vomits characterized for being a single episode of 500 ml or less.
By hemagglutinin protease (HA/P) is meant the protein secreted by V. cholerae manifesting dual function, being one of them the ability to agglutinate the erythrocytes of certain species and the other the property to degrade or to process proteins such as mucine and the cholera toxin.
By celA is meant the nucleotide sequence coding for the synthesis of the endoglucanase A. This protein naturally occurs in Clostridium thermocellum strains and has a β (1-4) glucan-glucano hidrolase activity able to degrade cellulose and its derivatives.
The term MSHA is referred to the structural fimbria of the surface of V. cholerae with capacity to agglutinate erythrocytes of different species and that is inhibited by mannose.
By reversion to virulence mediated by VGJΦ is meant the event in which a previously attenuated strain obtained by the suppression of CTXΦ genes reacquire all the genes of this phage through a mechanism completely dependent and mediated by VGJΦ and the interaction with its receptor, MSHA.
The possibility of disseminating the CTXΦ phage in a process mediated by VGJΦ is that in which the filamentous phage VGJΦ form a stable hybrid structure (HybPΦ) through genetic recombination with the DNA of CTXΦ and disseminate its genome with active genes toward other strains of V. cholerae, which could be environmental non pathogenic strains, vaccine strains or other from different species.
Second, information of the previous art:
Clinical cholera is an acute diarrheal disease that result from an oral infection with the bacterium V. cholerae. After more than 100 years of research in cholera there remains the need for an effective and safe vaccine against the illness. Since 1817 man has witnessed seven pandemics of cholera, the former six were caused by strains of the Classical biotype and the current seventh pandemic is characterized by the prevalence of strains belonging to El Tor biotype. Recently, beginning in January of 1991, this pandemic extended to South America, and caused more than 25 000 cases of cholera and over 2 000 deaths in Peru, Ecuador and Chile. By November 1992, a new serogroup of V. cholerae emerged in India and Bangladesh, the 0139, showing a great epidemic potential and generating great concern through the developing world. These recent experiences reinforce the need for effective cholera vaccines against the disease caused by V. cholerae of serogroups O1 (biotype El Tor) and O139.
Because convalescence to cholera is followed by an state of immunity lasting at least three years, much efforts in Vibrio cholerae vaccinology have been made to produce live attenuated cholera vaccines, that closely mimics the disease in its immunization properties after oral administration, but do not result reactogenic to the individuals ingesting them (diarrhea, vomiting, fever). Vaccines of this type involve deletion mutations of all toxin genes encoded by CTXΦ. For example, the suppression of the cholera toxin and other toxins genes encoded in the prophage CTXΦ is a compulsory genetic manipulation during the construction of a live vaccine candidate (see inventions of James B. Kaper, WO 91/18979 and John Mekalanos WO 9518633 of the years 1991 and 1995, respectively).
This kind of mutants have been proposed as one dose oral vaccines, and although substantially attenuated and able to generate a solid immune responses (Kaper J. B. and Levine M. Patentes U.S. Pat. Nos. 06,472,276 and 581,406). However, the main obstacle for the widespread use of those mutants has been the high level of adverse reactions they produce in vaccinees (Levine and cols., Infect. and Immun. Vol 56, No1, 1988).
Therefore, achieving enough degree of attenuation is the main problem to solve during the obtainment of live effective vaccines against cholera. There are at least three live vaccine candidates, which have shown acceptable levels of safety, i.e., enough degree of attenuation and strong immunogenic potential. They are V. cholerae CVD103HgR (Classical Biotype, serotype Inaba) (Richie E. and cols, Vaccine 18, (2000): 2399-2410.), V. cholerae Perú-15 (Biotype El Tor, serotype Inaba) (Cohen M., and cols. (2002) Infection and Immunity, Vol 70, Not. 4, pag 1965-1970) and V. cholerae 638 (Biotype El tor, serotype Ogawa) (Benítez J. A. and cols, (1999), Infection and Immunity. February; 67(2):539-45).
Strain CVD103HgR is the active antigenic component of a live oral vaccine against cholera licensed in several countries of the world, the strains Perú-15 and 638 are other two live vaccine candidates to be evaluated in field trials in a near future.
However, there is a second problem of importance to solve in those live attenuated vaccine candidates; one is the environmental safety, specially related with the possible reacquisition and dissemination of the cholera toxin genes by existent mechanisms of horizontal transfer of genetic information among bacteria. In accordance with this, the attenuated vaccine strains of V. cholerae, could potentially reacquire virulence genes out of the controlled conditions of the laboratory, in an infection event with CTXΦ phage (Waldor M. K. and J. J. Mekalanos, Science 272:1910-1914) coming from other vibrios and later on contribute to their dissemination. This process could become relevant during vaccination campaigns where people ingest thousands of millions of attenuated bacteria and keep shedding similar quantities in their stools during at least 5 days. Once in the environment, bacteria have the possibility of acquiring genetic material from other bacteria of the same or different species of the ecosystem. For these reasons, at present it is desirable to obtain vaccine candidates with certain characteristics that prevent or limit the acquisition and dissemination of CTXΦ, and especially of the genes coding for the cholera enterotoxin. As a consequence, this is the field of the present invention.
Bacterial viruses, known as bacteriophages, have an extraordinary potential for gene transfer between bacteria of the same or different species. That is the case of CTXΦ phage (Waldor M. K. and J. J. Mekalanos, 1996, Science 272:1910-1914,) in V. cholerae. CTXΦ the genes of carries the genes that encode cholera toxin in V. cholerae and enters to the bacteria through interaction with a type IV pili, termed TCP, from toxin co-regulated pilus. TCP is exposed on the external surface of the vibrios. In accordance with published results, under optimal laboratory conditions the CTXΦ phage reaches titers of 106 particles or less by ml of culture in the saturation phase; this allows classifying it as a moderately prolific bacteriophage. Equally the expression of the TCP receptor of this phage has restrictive conditions for its production. In spite of these limitations, the existence of this couple bacteriophage-receptor, limits in some way the best acceptance of live cholera vaccines, that is why depriving the bacteria from the portal of entrance to this phage is a desirable modification.
There are two theoretical ways of preventing the entrance of CTXΦ into V. cholerae, 1) suppressing the expression of TCP or 2) removing the TCP sites involved in phage receptor interaction. None of the two forms has been implemented due to the essentiality of TCP for proper colonization of the human intestine and elicitation of a protective immune response. It should be noted that sites involved in the TCP-CTXΦ interaction are also needed for the colonization process. (Taylor R. 2000. Molecular Microbiology, Vol (4), 896-910).
Several strategies that counteract the entrance of the virus have been evaluated such as preventing the integration of the phage to the bacterial chromosome and its stable inheritance, consisting in the suppression of the integration site and in the inactivation of recA gene to avoid recombination and integration to other sites of the chromosome. (Kenner and cols. 1995. J. Infect. Dis. 172:1126-1129).
Also, it has been recently described that the entry of CTXΦ into V. cholerae depends on the genes TolQRA, however this mutation produces sensitive phenotypes not undesired in vaccine candidates of cholera and it has not been implemented. (Heilpern and Waldor. 2000. J. Bact. 182:1739).
Further methods that prevent the entrance of phages carryings essential virulence determinants to cholera vaccine strains or other vaccine strains have not been described.